SUMMARY A constraint to studying human cancer is the limited availability of models with appropriate human stroma and immunity. This caveat is particularly relevant for melanoma, given the pivotal role the immune system plays in its development. To address these limitations, we plan to study melanoma biology and therapy in a humanized mouse (HM) model. We initially developed a mis-matched HM (mHM; tumor and hematopoietic stem cells [HSCs] from different sources), and now have generated an autologous HM (aHM; tumor, HSCs and mesenchymal stem cells [MSCs] from the same patient). In HM bearing tumors the human immune and mesenchymal cells from the patient's bone marrow homed into the growing tumor, migrated into the pre- existing mouse stroma, and interacted with the human cancer cells. Tumors grown in HM more closely resembled the originator tumor than those grown in non-HM mice, and the drift in gene expression caused by prior passaging was partially reversed. Signaling in key immune and stroma pathways was more prominent and closely resembled the originator patient in HM vs. non-HM models. The immune cells mounted effective tumor-specific immune responses, mediated by human immune cells including T cells. Significantly, immune responses upon immune therapies in the HM melanoma models showed a correlation with the patient's therapy results. Two salient and under-studied issues limit the wider application of HM: The relevance of the degree of tumor and immune matching, which can affect the faithfulness of immune response, and that can only be appreciated by comparing mHM and aHM. A second criticism to HM is xenogenic education of human T cells on mouse thymic cells, resulting from the lack of a functional human thymus in such models. In this application we will further address the relevance of an autologous versus mis-matched HM to test which approach gives rise to a more representative model of melanoma; secondly, using the same precursor cells from the patient we will reconstitute a functional thymus. The overarching goal of this proposal is to conduct a co-clinical trial where we will accomplish the following: 1) prospectively generate and characterize aHM and mHM from 20 melanoma patients, 2) test which approach yields a more representative melanoma HM model, 3) test in HM the immune drugs (cytotoxic T-lymphocyte-associated protein 4 [CTLA4] and programmed cell death protein 1 [PD-1] inhibitors) that each patient received and correlate with the clinical data, 4) identify the mechanisms involved in resistance to CTLA4/PD-1 inhibitors; and finally as an exploratory Aim in selected cases, we will 5) generate thymic epithelium from the same patient's HSCs that will result in HM with a fully autologous melanoma, thymus and immune system, thus enabling immune cell education in a strictly human context. This project will advance our understanding of the tumor-host interaction in melanoma and human cancer, by better characterizing the interplay between melanoma cells and the immune and stroma systems, leading to the discovery of new approaches to improve personalization of therapy and improve outcomes.